Comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris
- Boran Altincicek1Email author and
- Andreas Vilcinskas1
https://doi.org/10.1186/1742-9994-5-6
© Altincicek and Vilcinskas; licensee BioMed Central Ltd. 2008
Received: 01 April 2008
Accepted: 27 April 2008
Published: 27 April 2008
Abstract
Background
The planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris have emerged as valuable model organisms in regeneration and stem cell research because of their prominent ability to regenerate a complete organism from any small body fragment. Under natural conditions wounding may result from predator attacks. These injuries open their innermost to a wide array of microbes present in the environment. Therefore, we established the hypothesis that regeneration processes may be linked to or at least accompanied by innate immune responses. In order to screen for septic wounding inducible genes we dissected individuals using a scalpel in the presence of a crude bacterial lipopolysaccharide preparation that is commonly used to elicit innate immune responses in animals and applied the suppression subtractive hybridization technique that selectively amplifies cDNAs of differentially expressed genes.
Results
This analysis revealed the induced expression of 27 genes in immune challenged Schmidtea and 35 genes in immune challenged Hydra. Identified genes from both animals encode proteins that share sequence similarities with potential homologues from other organisms known to be involved in signaling (e.g. calreticulin in Schmidtea and major vault protein in Hydra), stress responses (e.g. Hsp20 in Schmidtea and a PRP19/PSO4 DNA repair protein in Hydra), or to represent potential antimicrobial effectors (e.g. perforin-like protein in Schmidtea and PR-1-like protein and neutrophil cytosolic factor 1 in Hydra). As expected, septic wounding also induces expression of genes in Schmidtea and Hydra potentially involved in tissue remodeling associated with regeneration processes (e.g. matrix metalloproteinase in Schmidtea and a potential von Willebrand factor in Hydra).
Conclusion
We identified numerous immune-inducible genes in Hydra and Schmidtea that show a similar distribution corresponding to their physiological roles, although lineages of both animals split from their common ancestor for more than five hundred millions of years. The present study is the first analysis of immune-inducible genes of these two phylogenetically distant model organisms of regeneration and provide numerous candidate genes that we can use as a starting point for comparative examination of interrelationships between immunity and homeostasis.
Keywords
Background
To date, there is no satisfactory answer to the question why some animals have higher regeneration capacities than others. The ability to replace lost or injured body parts is widely distributed among animals, whereas regeneration of a complete organism from any small body fragment is restricted to only few animal phyla and is accompanied by the ability to reproduce asexually by budding or fission [1, 2]. These features have been attributed to a stable population of stem cells known as neoblasts in Schmidtea mediterranea [3, 4] and to both stem cell-based mechanisms and transdifferentiation in Hydra vulgaris [5, 6]. These two phylogenetically distant animals with remarkable regeneration capacities attract renewed attention as powerful model organisms since both, S. mediterranea and H. vulgaris, are amenable to systemic RNAi mediated gene silencing and other genetic tools for functional gene analyses [7–11].
Phyla distribution of animals whose immune-inducible genes have been analyzed by the SSH method. A major evolutionary divide occurs in the animal kingdom between the so-called radially symmetric animals, which includes the cnidarian Hydra, and the bilaterally symmetric animals, which includes Schmidtea. The phylogenetic position of these two model organisms that have been selected for investigations in the present work (indicated by red color) promotes comparative analysis that may help to reconstruct functions and evolution of genes involved in regeneration and immunity. Previously, Branchiostoma belcheri [23], Danio rerio [24], Platynereis dumerilii [19], Biomphalaria glabrata [20,21], Thermobia domestica [12], Tribolium castaneum [14], Eristalis tenax [13], Manduca sexta [17], and Galleria mellonella [15] have been analyzed by the SSH method.
In this study, we report profiles of injury-inducible genes from Schmidtea and Hydra, including genes that encode proteins potentially involved in e.g. immune-inducible signaling and defense reactions. Comparisons of potential calreticulin, matrix metalloproteinase (MMP), and perforin with homologues from other organisms provide novel insights into the ancestral complexity and evolution of the metazoan immune system.
Results and Discussion
Subtracted cDNA libraries of immune challenged S. mediterranea and H. vulgaris
Distribution of immune-inducible transcripts identified by the SSH approach in Schmidtea and Hydra. Septic injury results in an immune-induced expression of totally 27 genes in S. mediterranea (A) and 35 genes in H. vulgaris (B) as obtained by the SSH approach. Interestingly, the genes have a similar distribution regarding their physiological roles.
cDNAs from the subtracted S. mediterranea library.
Cluster | GenBank Accession No. of EST | Highest BlastX match | E value | PFAM/InterPro |
---|---|---|---|---|
Defense protein | ||||
FC823226 | AAR82935: macrophage expressed protein (Haliotis corrugata) | 3e-22 | Membrane attack complex component/perforin/C9 | |
Signaling proteins | ||||
FC823227 | XP_001119958: similar to p21/Cdc42/Rac1-activated kinase 1 (Apis mellifera) | 2e-35 | P21-activated kinase | |
FC823228 | XP_972570: similar to PhosphoLipase C family member (plc-1) (Tribolium castaneum) | 8e-09 | ||
FC823229 | NP_524378: sarah CG6072-PA (Drosophila melanogaster) | 5e-23 | Calcipressin | |
FC823230 | CAK04334: similar to human chromodomain helicase DNA binding protein 2 (CHD2) (Danio rerio) | 6e-53 | DEAD-like helicases, N-terminal | |
FC823231 | XP_001197550: similar to mFLJ00175 protein, partial (Strongylocentrotus purpuratus) | 2e-72 | C2 calcium-dependent membrane targeting | |
FC823232 | XP_392689: similar to Calreticulin CG9429-PA (Apis mellifera) | 8e-99 | Calreticulin | |
Stress related proteins | ||||
FC823233 | AAW25328: SJCHGC09367 protein (Schistosoma japonicum) | 7e-07 | Hsp20/alpha crystallin family | |
FC823234 | XP_696859: hypothetical protein (Danio rerio) | 7e-27 | RAD50 | |
Cell adhesion related proteins | ||||
FC823235 | CAA37667: pre-pro-hatching enzyme (Paracentrotus lividus) | 4e-20 | Peptidase_M10 | |
FC823236 | AAX25986: SJCHGC09378 protein (Schistosoma japonicum) | 3e-52 | Collagen triple helix repeat | |
FC823237 | ZP_01611880: putative adhesion lipoprotein (Alteromonadales bacterium TW-7) | 5e-02 | Beta-Ig-H3/fasciclin | |
FC823238 | AAX25800: SJCHGC02149 protein (Schistosoma japonicum) | 4e-06 | Cell adhesion molecule | |
Cytoskeleton proteins | ||||
FC823239 | XP_419249: hypothetical protein (Gallus gallus) | 2e-64 | Tubulin/FtsZ | |
FC823240 | XP_001176242: similar to cytoskeletal actin CyIIb (Strongylocentrotus purpuratus) | 3e-38 | Actin | |
FC823241 | XP_624678: similar to Myosin IA (MIA) (Brush border myosin IA) (BBMIA) (Apis mellifera) | 8e-61 | IQ calmodulin-binding region; Myosin_head | |
Metabolism | ||||
FC823242 | AAB53748: 3-hydroxy-3-methylglutaryl-CoA reductase (Oryza sativa) | 1e-14 | Hydroxymethylglutaryl-coenzyme A reductase | |
FC823243 | CAF95654: unnamed protein product (Tetraodon nigroviridis) | 2e-21 | Selenophosphate synthetase | |
FC823244 | AAU03135: glutamate dehydrogenase (Hylobates lar) | 1e-55 | Glu/Leu/Phe/Val dehydrogenase | |
FC823245 | XP_001177631: similar to Gldc-prov protein (Strongylocentrotus purpuratus) | 4e-52 | Glycine cleavage system P-protein | |
Protein biosynthesis | ||||
FC823246 | AAL49199: RE63456p (Drosophila melanogaster) | 9e-18 | Ribosomal_L34e | |
Unknown proteins | ||||
FC823247 | CAE65027: Hypothetical protein CBG09865 (Caenorhabditis briggsae) | 1e-03 | ||
FC823248 | Hypothetical protein | NSM1 | Ubiquitin | |
FC823249 | Hypothetical protein | NSM | ||
FC823250 | Hypothetical protein | NSM | ||
FC823251 | Hypothetical protein | NSM | ||
FC823252 | Hypothetical protein | NSM |
cDNAs from the subtracted H. vulgaris library.
Cluster | GenBank Accession No. of EST | Highest BlastX match | E value | PFAM/InterPro |
---|---|---|---|---|
Defense proteins | ||||
FC823190 | XP_001356627: GA14264-PA (Drosophila pseudoobscura) | 5e-07 | SCP/Tpx-1/Ag5/PR-1/Sc7 family of extracellular domains | |
FC823191 | XP_001063612: similar to neutrophil cytosolic factor 1 (Rattus norvegicus) | 7e-35 | p47-phox (neutrophil cytosolic factor 1) | |
Signaling proteins | ||||
FC823192 | XP_690149: similar to diacylglycerol kinase eta2 (Danio rerio) | 4e-18 | ||
FC823193 | XP_001201815: hypothetical protein, partial (Strongylocentrotus purpuratus) | 6e-96 | Major vault protein, N-terminal | |
FC823194 | XP_001489489: similar to THO complex 4 (Equus caballus) | 6e-06 | ||
FC823195 | AAZ99727: dickkopf-like protein Dlp-1 precursor (Hydra magnipapillata) | 4e-44 | ||
FC823197 | XP_421629: similar to Transmembrane 9 superfamily protein member 3 precursor (Gallus gallus) | 2e-79 | Nonaspanin (TM9SF) | |
FC823198 | Q6NZZ4: Transmembrane protein 107 (Danio rerio) | 3e-21 | ||
Stress related proteins | ||||
FC823199 | XP_867913: similar to PRP19/PSO4 homologue (Canis familiaris) | 1e-91 | WD40/YVTN repeat-like | |
FC823200 | AAM76192: LD36241p (Scythe protein) (Drosophila melanogaster) | 5e-03 | ||
Cell adhesion related proteins | ||||
FC823201 | AAT37632: von Willebrand factor (Branchiostoma belcheri tsingtaunese) | 4e-11 | ||
Cytoskeleton proteins | ||||
FC823202 | EAW96898: myosin, light polypeptide 6B, (Homo sapiens) | 1e-10 | Calcium-binding EF-hand | |
FC823203 | CAA48796: actin (Podocoryne carnea) | 3e-116 | Actin/actin-like | |
FC823204 | P18320: Profilin (Anthocidaris crassispina) | 5e-41 | Profilin/allergen | |
FC823205 | P34032: Thymosin beta-4 (Oryctolagus cuniculus) | 6e-05 | Thymosin beta-4 | |
FC823206 | CAM37732: flagellar calcium-binding protein, putative (Leishmania braziliensis) | 7e-16 | Calcium-binding EF-hand | |
Metabolism | ||||
FC823207 | XP_001366187: similar to NADH dehydrogenase (Monodelphis domestica) | 6e-02 | NADH dehydrogenase | |
FC823208 | XP_395823: similar to Phosphomannomutase (Apis mellifera) | 1e-25 | Phosphomannomutase | |
FC823209 | XP_001197762: similar to NAD(P)H:quinone oxidoreductase (Strongylocentrotus purpuratus) | 1e-15 | Oxidoreductase | |
FC823210 | XP_001494492: hypothetical protein (Equus caballus) | 3e-15 | MoeA, N-terminal, domain I and II | |
FC823211 | XP_783197 hypothetical protein (Strongylocentrotus purpuratus) | 3e-24 | Methyltransferase type 11 | |
FC823212 | NP_001017016: exosome component 6 (Xenopus tropicalis) | 8e-05 | Exonuclease, RNase T and DNA polymerase III | |
FC823213 | NP_957038: small fragment nuclease (Danio rerio) | 3e-50 | Ribonuclease PH related | |
FC823214 | NP_990451: enolase 1 (Gallus gallus) | 3e-99 | Enolase | |
FC823215 | ABC25045: mitochondrial succinate dehydrogenase flavoprotein subunit (Hydra vulgaris) | 4e-94 | Fumarate reductase/succinate dehydrogenase flavoprotein, | |
Protein biosynthesis | ||||
FC823216 | AAR09792: similar to Drosophila melanogaster eIF-5A (Drosophila yakuba) | 2e-06 | eIF-5A | |
FC823217 | NP_001016714: ribosomal protein L28 (Xenopus tropicalis) | 8e-15 | Ribosomal protein L28e | |
FC823218 | AAY43411: ribosomal protein L23 (Phytophthora infestans) | 4e-34 | Ribosomal protein L14b/L23e | |
FC823219 | XP_392565: similar to Ribosomal protein L18 (Apis mellifera) | 4e-60 | Ribosomal protein L18e | |
FC823220 | AAY97868: cytoplasmic ribosomal protein S13 (Lycopersicon esculentum) | 1e-47 | Ribosomal protein S15 | |
Unknown proteins | ||||
FC823221 | XP_413837: hypothetical protein (Gallus gallus) | 6e-02 | ||
FC823222 | ZP_01629577: hypothetical protein (Nodularia spumigena CCY9414) | 9e-02 | ||
FC823223 | Hypothetical protein | NSM1 | ||
FC823224 | Hypothetical protein | NSM | ||
FC823225 | Hypothetical protein | NSM |
Signaling
In animals, hereditable receptors including the prominent Toll-receptors recognize damage- or pathogen-associated molecular pattern molecules and engage multiple immune-related signaling pathways [27]. Here, we identified a Schmidtea cDNA encoding a protein that exhibits highest sequence similarities to p21/Cdc42/Rac1-activated kinase 1 from Apis. In mammals, this kinase is believed to act directly on the JNK (c-Jun N-terminal kinase) MAP (mitogen-activated protein) kinase pathway [28]. JNK is a prominent stress kinase that has been studied mostly in the context of cellular stress and apoptotic cell death following, for example, heat shock, DNA damage, and inflammation [29].
Calcium signals in human immune cells participate in the regulation of cell differentiation and influence lymphocyte motility, immunological synapse formation, degranulation and phagocytosis [30]. In agreement, we found several predicted proteins in Schmidtea and Hydra that show similarities to members of the calcium signaling pathways suggesting that calcium pathways may be important in immune responses in these animals. One Schmidtea protein with highest similarities to ferlin family proteins that are known to be associated with both plasma and nuclear membranes contains a C2 domain that may play a role in calcium-mediated membrane fusion events during membrane regeneration and repair [31]. Additionally, we identified a Schmidtea cDNA that encodes a potential calcipressin homologue. Vertebrate calcipressins modulate the pattern of calcineurin-dependent transcription, and may influence calcineurin activity beyond calcium to integrate a broad array of signals into the cellular response [32]. The importance of calcineurin in immunity is highlighted by the use of calcineurin inhibitors such as cyclosporine as prominent immunosuppressive drugs in humans [33]. Furthermore, we found a potential Schmidtea phospholipase C that may generate inositol triphosphate and diacylglycerol by hydrolyzing phosphatidylinositol which in turn leads to raising the level of intracellular calcium. In Hydra, we determined the induced expression of a potential diacylglycerol kinase that balances between two signaling lipids, diacylglycerol and phosphatidic acid, by phosphorylating the former one. Therefore, diacylglycerol kinases may be involved in numerous lipid and calcium signaling pathways on transient or continuous demands, respectively [34].
Phylogenetic analysis of Schmidtea calreticulin and homologues from selected species. We identified a calreticulin protein in the Schmidtea cDNA library. Using corresponding EST sequence and further EST sequences from the public database (DN316126 and EG417940) we constructed the potential full-length protein. A Bayesian protein tree was generated using calreticulin sequences from Schmidtea and from selected species (Trypanosoma cruzi, EAN82340; Leishmania infantum, XP_001467584; Dictyostelium discoideum, XP_639010; Eisenia fetida, ABI74618; Schistosoma japonicum, ABK34456; Taenia solium, AAK52725; Echinococcus granulosus, AAX73173; Apis mellifera, XP_392689; Drosophila melanogaster, NP_524293; Tribolium castaneum, XP_971763; Crassostrea gigas, BAF63639; Onchocerca volvulus, AAA59056; Necator americanus, CAA07254; Heligmosomoides polygyrus, CAL30086; Caenorhabditis elegans, NP_504575; Ixodes scapularis, AAT99573; Homo sapiens, NP_004334; Strongylocentrotus purpuratus, NP_999643). As outgroup we selected plant Arabidopsis thaliana calreticulin (NP_001031199). This phylogenetic analysis reveal that calreticulins are evolutionarily conserved and that Schmidtea calreticulin (indicated by gray shading) form an own group with calreticulins from parasitic Platyhelminthes. The scale bar represents the substitutions per site.
Defense molecules
A potential perforin exist in Schmidtea that is related to other known perforins. We identified a perforin-like protein in the Schmidtea cDNA library. Using corresponding EST sequence and further EST sequences from the public database (DN313739, EG343971, DN299412, DN307006) we constructed the potential full-length protein. The alignment with perforin sequences from Haliotis corrugata (AAR82935), Homo sapiens (AAI12231), Danio rerio (XP_688214), and the sponge Suberites domuncula (CAI68018) reveal that perforins are conserved in evolution. Red color indicates 90% consensus and blue color 40% consensus.
A potential antimicrobial peptide in Hydra. We identified a Hydra cDNA (FC823225) that encodes a cationic peptide with predicted α-helical structure [67]. It shares significant similarities with antimicrobial esculentin-1B (P40844) from Rana esculenta.
Interestingly, one immune-inducible transcript from Hydra exhibits significant sequence similarities to major vault proteins from other organisms. Very recently, major vault protein were found to be rapidly recruited to lipid rafts when human lung epithelial cells are infected with Pseudomonas aeruginosa. Major vault protein has been demonstrated to be essential for optimal epithelial cell internalization and clearance of P. aeruginosa indicating that it makes a substantial contribution to epithelial cell-mediated resistance to infection in mammals [42] and probably also in Cnidaria. However, the group of Thomas C.G. Bosch has recently described that in Hydra, similar as in humans, the immune system maintains a substantial resident beneficial microbiota on their epithelia [43]. This suggests that Hydra is able to discriminate 'friend' from 'foe' by killing entities that do damage and let those live that are commensals or mutualists which is in agreement to Poly Matzingers' proposed danger model of mammalian immunity [44] and our recent findings from insects that endogenous alarm signals induce innate immune responses during infection [45, 46].
Cellular homeostasis, cell adhesion related proteins, and regeneration
An identified Schmidtea MMP gene is related to Hydra MMP and to MMPs from insects and mammals. (A) All known MMPs from the fully genome sequenced ecdysozoan Drosophila melanogaster and humans (Deuterostomia) were aligned with the Schmidtea-MMP (AY068367) and the active site sequences are shown (MMP active site consensus sequence: HEXGHXXGXXHS). (B) A Bayesian protein tree was generated and we found that the Schmidtea-MMP grouped together with the MMP from H. vulgaris (AAD45804) nearest to MMP-1 from D. melanogaster. UniProt accession numbers for MMPs are: Drosophila1, Q9GTK3; Drosophila2, Q8MPP3; Human19, Q99542; Human28, Q9H239; Human11, P24347; Human21, Q8N119; Human17, Q9ULZ9; Human25, Q9NPA2; Human14, P50281; Human15, P51511; Human16, P51512; Human24, Q9Y5R2; Human20, O60882; Human12, P39900; Human13, P45452; Human1, P03956; Human8, P22894; Human3, P08254; Human10, P09238; Human7, P09237; Human26, Q9NRE1; Human23, O75900; Human2, P08253; Human9, P14780. Posterior probabilities are plotted at nodes. The scale bar represents the substitutions per site.
In addition, we found a Hydra protein with highest similarities to vWF (von Willebrand factor) proteins from other animals. This vWF protein is crucial for vertebrate blood clotting by binding to platelet receptors and collagens [55] and, in Hydra, may be important for cell-cell or cell-basement membrane adhesion processes.
Quantitative real time RT-PCR analysis of selected Schmidtea genes that are up-regulated in response to septic wounding. The mRNA levels of selected genes in immune challenged animals (gray bars) were determined and are shown relative to their expression levels in untreated animals (black bars). The expression of the house-keeping gene 18S rRNA was not significantly influenced by the treatment. Results represent mean values of three independent determinations ± SD. Statistically significant differences were determined using Student's t-test and are indicated by asterisks (p < 0.05) or the determined p values were inserted.
Conclusion
Here, we report a comparative analysis of immune-inducible genes in two phylogenetically distant model organisms of regeneration and stem cell research. We selected the cnidarian H. vulgaris and the planarian S. mediterranea for targeted screening of genes that are up-regulated upon large-scale septic wounding using the SSH method. Obtained results highlight the ancient origin of some genes (e.g. MMPs, calreticulin, p47-phox, perforin, and major vault protein) known from vertebrate immunity. This is consistent, for instance, with the findings that even the most ancient animals, porifera, possess functional Toll-like receptors [56] and with the recent report of a bioinformatic analysis of the immune repertoire in Cnidaria [39]. However, screening for immunity-related genes in genomic or EST databases only allows identification of genes that share sequence similarities with known genes, while the SSH-approach is particularly suitable for the experimental screen for yet unknown or unexpected genes and, therefore, complement the bioinformatic approaches. Confirming this, the present study increases the number of genes identified in Schmidtea and Hydra (e.g. phospholipase C and perforin for S. mediterranea and major vault protein and vWF protein in H. vulgaris). In addition, using the SSH-method we have recently identified novel insect antimicrobial peptides among which some of them emerged as promising templates for the rational design of second generation antibiotics [57] or as transgenes for the generation of disease-resistant crops [58]. Hence, identified immunity-related genes from Schmidtea and Hydra may have also potential therapeutic value. Additionally, it will be a challenge to elucidate physiological functions of the presently identified genes during immune responses and homeostasis and to employ Schmidtea and Hydra as model organisms for the investigation of molecular interactions of pathogens with the host innate immune system.
Methods
Immune challenge of Schmidtea mediterranea and Hydra vulgaris and RNA isolation
The asexual strain of S. mediterranea (originated from a fountain in Montjuïc, Barcelona, Spain) was kept at 18°C in darkness and fed once per week with sheep liver. One-week-starved about 7-mm-long animals were used for experiments. H. vulgaris (Zürich strain, originally isolated by P. Tardent) was cultured at 18°C as described [59]. Septic wounding was performed by dissecting animals in two parts using a scalpel in the presence of 50 μg/ml LPS (purified Escherichia coli endotoxin 0111:B4, Cat. No.: L2630, Sigma, Taufkirchen, Germany). Total RNA was extracted from 14 h post immune challenged animals using the TriReagent isolation reagent (Molecular Research Centre, Cincinnati, Ohio, USA) according to the instructions of the manufacturer. RNA integrity was confirmed by ethidium bromide gel staining and quantities were determined spectrophotometrically.
Construction of subtracted cDNA libraries using the SSH method
In order to identify genes that are differentially expressed in response to septic injury we performed the suppression subtractive hybridization method using RNAs from immune challenged and untreated S. mediterranea and H. vulgaris, respectively, the SMART PCR cDNA synthesis Kit (Clontech, Mountain View, CA, USA), and the PCR-Select cDNA subtraction Kit (Clontech), according to the protocols of the manufacturer. Colony PCR of each 288 randomly picked colonies and blot hybridization have been performed similar as described recently [19].
Sequencing and computer analysis of cDNA sequence data
Plasmid isolation of positively screened colonies was performed with the FastPlasmid Mini Kit (Eppendorf, Hamburg, Germany) and purified plasmids were custom sequenced by Macrogen Inc. (Seoul, South-Korea). Blast [60] was used to identify corresponding gene sequences in the public sequence databases. InterProScan [61] was used for an integrated search in PROSITE, Pfam, and PRINTS databases at EMBL-European Bioinformatics Institute and to predict signal sequences and transmembrane regions.
Sequence alignments and phylogenetic analysis
Multiple sequence alignments were computed using blosum62 program [62]. For phylogenetic reconstruction, we used the software package MrBayes 3.1.2 [63] which combines Bayesian inference and Markov chain Monte Carlo convergence acceleration techniques known as Metropolis coupling. The best fixed-rate model of amino acid evolution was determined by model jumping among nine possible models. The model with the overall highest posterior probability was WAG model [64] for the MMPs after 106 generations and for calreticulins after 2 × 106 generations. We used convergence diagnostic (i.e., the standard deviation of split frequencies) to determine whether the run length is sufficient. The average standard deviation of split frequencies was 0.0051 for MMPs and 0.0023 for the calreticulins. This indicated that the two chains that were run converged on similar results in all cases. The 50% majority rule tree presented here was constructed from all sampled trees with the first 25% of all trees ignored as burn in. Posterior probabilities plotted at the nodes can be interpreted as the probability that the tree or clade is correct [65].
Quantitative real-time PCR
Genes analyzed by quantitative real time RT-PCR.
Gene | accession number | Forward primer 5'-3' | Reverse primer 5'-3' |
---|---|---|---|
18S rRNA | EG412600 | ATGGTTGCAAAGCTGAAACT | TCCCGTGTTGAGTCAAATTA |
HMG | EE666986 | CGCATCTGATCCATGCAAGC | TGTACCTCCCCCGATTGTTCC |
Calreticulin | EG417940 | CCCTTGGGTGCATCCAGAAA | CGGACTTCACCTGCCACAGA |
Hsp20 | DN302574 | GGAATCTGGGGTGAGCTTGG | CGGTTGATGGCTCAATGCAC |
MMP | AY068367 | TCGGCTTCTGGTTCCGATGT | CGGGCAAAAGCTGCAGAACT |
Perforin | DN307006 | GGCTGCCTGCATCTGTTTGA | TTGAGCCGTAGTCCGCCAAT |
Tubulin | DN315207 | TCCGGGTGGAGATTTGGCTA | GCACGCTTGGCATACATTAGGTC |
Actin | EC386294 | ATCCTGGCATTGCTGATCGT | TGGGGGAGCAACAATCTTGA |
Declarations
Acknowledgements
We thank Meike Fischer for excellent technical assistance and Katja Altincicek for critical reading of the manuscript. We are grateful to Emili Saló (University of Barcelona, Spain) and Luca Gentile (Max Planck Institute for Molecular Biomedicine, Münster, Germany) for kindly providing us with Schmidtea mediterranea and Monika Hassel (Philipps University of Marburg, Germany) for kindly providing us with Hydra vulgaris.
Authors’ Affiliations
References
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